Summary The long term goal of this research is to utilize toll-like receptor 5 (TLR5) agonist based immunotherapy with immunomodulatory radiation treatment in the development of a safe and effective combinatorial approach to control liver metastatic disease. The objective of this project is to demonstrate improved therapeutic efficacy against liver metastatic disease using this novel treatment approach and to provide a mechanistic foundation to advance further development of the combined strategy. The hypothesis underlying this research is that TLR5 agonist mediated immune response in the normal liver microenvironment combined with the immunomodulatory effects of liver radiation treatment (RT) generate potent antitumor immunity to eliminate liver metastases. This theory is based on recent cumulative data indicating that the liver microenvironment possesses a unique character in which TLR5 agonists elicit a significant antitumor immune response mediated by TLR5 responsive hepatocytes and on pilot experiments illustrating that the immune stimulatory effects of ionizing radiation applied to the liver enhance and extend the therapeutic reach of TLR5 agonist immunotherapy against liver metastases. In this project, the hypothesis will be tested using the highly aggressive murine colorectal cancer and uveal melanoma liver metastasis models and the newly generated and pharmacologically optimized TLR5 agonist, entolimod. The research plan includes testing different combination therapy regimens and comparing the results of their application to that of liver radiation and TLR5 stimulation individually for antitumor efficacy against liver metastatic growth using the experimental colorectal cancer and spontaneous uveal melanoma liver metastasis models (Aim 1). Single dose and fractionated RT will be applied at different times (adjuvant and neoadjuvant) relative to systemic entolimod administration to determine the most efficacious regimens of the combined therapy. The role of the immunological changes in the hepatic microenvironment in the development of an efficacious antitumor immune response will be determined using phenotypic and functional assays (Aim 2). The results will further the development of a clinical protocol for this innovative approach. While the murine colorectal cancer and uveal melanoma liver metastasis models were chosen due to the significant medical need, clear translational path and encouraging preliminary results obtained with these models, the results will provide information relevant to other cancers with a high propensity to metastasize to the liver. Completion of this project will provide supporting pre-clinical efficacy data to facilitate advancement to clinical trials and a mechanistic foundation on which to base further investigation of the molecular mechanisms responsible for the antitumor activity of this innovative therapeutic approach that is critically needed against liver metastasis.